Peroxicats consortium at the kick-off meeting in Madrid.

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Fernandez-Fueyo E, Ruiz-Dueñas FJ, Miki Y, Martínez MJ, Hammel KE, Martínez AT (2012)
J. Biol. Chem., 287. 16903-16916.


The white-rot fungus Ceriporiopsis subvermispora delignifies lignocellulose with high selectivity, but until now has appeared to lack the specialized peroxidases, termed lignin peroxidases (LiPs) and versatile peroxidases (VPs), that are generally thought important for ligninolysis. We screened the recently sequenced C. subvermispora genome for genes that encode peroxidases with a potential ligninolytic role. A total of 26 peroxidase genes was apparent after a structural-functional classification based on homology modeling and a search for diagnostic catalytic amino acid residues. In addition to revealing the presence of nine heme-thiolate peroxidase superfamily members and the unexpected absence of the dye-decolorizing peroxidase superfamily, the search showed that the C. subvermispora genome encodes 16 Class II enzymes in the plant-fungal-bacterial peroxidase superfamily, where LiPs and VPs are classified. The 16 encoded enzymes include 13 putative manganese peroxidases and one generic peroxidase, but most notably two peroxidases containing the catalytic tryptophan characteristic of LiPs and VPs. We expressed these two enzymes in Escherichia coli and determined their substrate specificities on typical LiP/VP substrates, including nonphenolic lignin model monomers and dimers, as well as synthetic lignin. The results show that the two newly discovered C. subvermispora peroxidases are functionally competent LiPs, while also suggesting that they are phylogenetically and catalytically intermediate between classical LiPs and VPs. These results offer new insight into selective lignin degradation by C. subvermispora.

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Fernandez-Fueyo E, Ruiz-Dueñas FJ, Ferreira P, .... , Martínez AT, Vicuña R, Cullen D (2012)
Proc. Natl. Acad. Sci. USA, doi: 10.1073/pnas.1119912109.


Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn²⁺. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

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Hernández-Ortega A, Ferreira P, Merino P, Medina M, Guallar V, Martínez AT (2012)
ChemBioChem, 13. 427-435.


Primary alcohol oxidation by aryl-alcohol oxidase (AAO), a flavoenzyme providing H₂O₂ to ligninolytic peroxidases, is produced by concerted proton and hydride transfers, as shown by substrate and solvent kinetic isotope effects (KIEs). Interestingly, when the reaction was investigated with synthesized (R)- and (S)-α-deuterated p-methoxybenzyl alcohol, a primary KIE (≈6) was observed only for the Renantiomer, revealing that the hydride transfer is highly stereoselective. Docking of p-methoxybenzyl alcohol at the buried crystal active site, together with QM/MM calculations, showed that this stereoselectivity is due to the position of the hydride- and proton-receiving atoms (flavin N5 and His502 Nε, respectively) relative to the alcohol Cα-substituents, and to the concerted nature of transfer (the pro-S orientation corresponding to a 6 kcal mol⁻¹ penalty with respect to the pro-R orientation). The role of His502 is supported by the lower activity (by three orders of magnitude) of the H502A variant. The above stereoselectivity was also observed, although activities were much lower, in AAO reactions with secondary aryl alcohols (over 98 % excess of the R enantiomer after treatment of racemic 1-(p-methoxyphenyl)ethanol, as shown by chiral HPLC) and especially with use of the F501A variant. This variant has an enlarged active site that allow better accommodation of the α-substituents, resulting in higher stereoselectivity (S/R ratios) than is seen with AAO. High enantioselectivity in a member of the GMC oxidoreductase superfamily is reported for the first time, and shows the potential for engineering of AAO for deracemization purposes.

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Kluge M, Ullrich R, Scheibner K, Hofrichter M (2012)
Green Chem., doi: 10.1039/C1GC16173C.


Here we report on the stereoselective benzylic hydroxylation and C1–C2 epoxidation of alkylbenzenes and styrene derivatives, respectively, by a heme-thiolate peroxygenase (EC 1.11.2.1) from the fungus Agrocybe aegerita. Benzylic hydroxylation led exclusively to the (R)-1-phenylalkanols. For (R)-1-phenylethanol, (R)-1-phenylpropanol and (R)-1-tetralol, the ee reached >99%. For longer chain lengths, the enantiomeric excesses (ee) and total turnover numbers (TTN) decreased while the number of by-products, e.g. 1-phenylketones, increased. Epoxidation of straight chain and cyclic styrene derivatives gave a heterogeneous picture and resulted in moderate to excellent ee values and TTN: e.g., in the case of (1R,2S)-cis-β-methylstyrene oxide formation, an ee >99% and a TTN of 110000 was achieved. Hydroxylation and epoxidation were true peroxygenations, which was demonstrated by the incorporation of ¹⁸O from H₂¹⁸O₂ into the products. The use of fed-batch devices and varying feeding strategies for the substrate and co-substrate turned out to be a suitable approach to optimize peroxygenase catalysis.

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